Less is More: Design of a Highly Stable Disulfide-Deleted Mutant of Analgesic Cyclic α-Conotoxin Vc1.1.

Rilei Yu,Victoria A. L. Seymour,David J. Adams,Muharrem Akcan,David J. Craik,Quentin Kaas,Xinying Jia,Géza Berecki

doi:10.1038/srep13264

Abstract

Cyclic α-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H,C8F]cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 μM and rat N-type (Cav2.2) and recombinant human Cav2.3 calcium channels via GABAB receptor activation, with an IC50 of ~900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis.

Highlights

Conotoxins are disulfide rich peptide toxins produced by marine cone snail belonging to the Conus genus1–5. α -Conotoxins are a subgroup of conotoxins characterized by their ability to inhibit nicotinic acetylcholine receptors[4,5,6]
Molecular dynamics simulations over 30 ns were performed for the two variants that have a pair of hemi-cystine residues replaced by alanines
The conformation of [C3A,C16A]cVc1.1 deviated from the NMR solution structure of cVc1.1 over the course of the simulation, with the Cα root-mean-square deviation (RMSD) between core regions of the mutant peptide and cVc1.1 on average 1.5 Å

Summary

Introduction

Conotoxins are disulfide rich peptide toxins produced by marine cone snail belonging to the Conus genus1–5. α -Conotoxins are a subgroup of conotoxins characterized by their ability to inhibit nicotinic acetylcholine receptors (nAChRs)[4,5,6]. An orally active cyclic Vc1.1 (cVc1.1, Fig. 1) was engineered by joining the N- and C-termini of the peptide without affecting the three-dimensional structure or biological activity[9] This molecule was designed because a major obstacle generally impeding the use of bioactive peptides as drugs is their high susceptibility to enzymatic degradation[15,16]. The formation of multiple isomers complicates synthesis procedures and significantly increases the cost of production of peptides It has been shown for other disulfide-rich conotoxins that only selected disulfide bonds are crucial for stability and activity[21,22,23,24,25]. Crude cVc1.1 folds into two isomers in a 72:28 ratio[9], whereas [C2H,C8F]cVc1.1 forms only one isomer, gaining an immediate improvement of 28% in folding yield

Methods

Results

Discussion

Conclusion